Novel Mechanisms Underlying Alpha_2A Adrenergic Receptor-Cx43 Cellular Signaling Pathway Mediating Mirror Image Pain

Nowadays, Pain has been regarded extensively as one of serious diseases to human health. Pain could occur not only at the ipsilateral injury side but also at the same somites contralateral side to noxious stimuli. We term the later “Mirror image pain”. So far, the understand of persistence, chronicalization, diversities, personalization of mirror image pain is still difficult, although more and more rapid development of pain researches and controls, novel discovery of anti-pain drugs and strategies have been found.Many evidences have been demonstrated the alpha2 A adrenergic receptor(alpha2A-AR), gap junction intercellular communication, glia may participate in the regulation of pain through different molecular and cellular mechnisams. However, whether their interactions are underlying or not remains to be a necessary. In this paper, based on our well-established the experimental animal mirror image pain model mimicking natural scorpion sting inducing pain, the single side of rat hindpaw plantar subcutaneous injection of specific sodium channel activator Bm K I, one of main algogenic peptidergic components from the venom of Chinese scorpion Buthus Martensi Karsch(Bm K), was applied as pain-evoking stimulus. Combining with behavioral observation, real-time quantitative PCR, immunoblotting, immunofluorescence, live cell imaging and other experimental techniques, the dynamic expression in peripheral and central alpha2A-AR, connexin 43(Cx43), glia cells were detected to build novel alpha2A-AR- Cx43 signaling pathways regulating mirror image pain. The main research contexts and results were as follows:(i) The synergetic enhancement of inhibition of Bm K I-induced pain by Alpha2A-AR agonist and gap junction blockersThe unilateral plantar subcutaneous injection of Bm K I could provoke long term persistent spontaneous pain, thermal hyperalgesia and bilateral mirror mechanical V hyperalgesia. Pre-treatment with intrathecal administration of alpha2A-AR agonist, clonidine, or gap junction blocker carbenoxolone(CBX) could significantly inhibit Bm K I induced-pain behaviors. Moreover, the effects of clonidine were enhanced strongly by CBX when injected combinedly. These results demonstrate that the spinal and dorsal root ganglia(DRG) alpha2A-AR and Cx 43 are involved in Bm K I induced-pain, implying that alpha2A-AR- Cx 43 signaling pathway may underlie the induction, development and maintenance of mirror image pain.(ii) The differential expression of dorsal root ganglion alpha2A-AR-Cx43 and mirror image painq RT-PCR and Western blotting results showed that Bm K I could induce up-regulation of alpha2A-AR m RNA, satellite glia marker protein GFAP and Iba1, but down-regulation of Cx43 in rat bilateral DRG. Moreover, immunofluorescent detection demonstrated that alpha2A-AR co-localized distinctly with Cx43, GFAP, OX42, Iba1, large-diameter neuron marker protein NF200 and IB4. The co-localization of alpha2A-AR with these cellular markers was elevated after Bm K I injection. On the contrary, the expression of alpha2A-AR-Cx43 itself and co-localization with other marker proteins was significantly reversed by pre-treatment with intrathecal clonidine, an alpha2A-AR agonist. These results suggest that dynamic and diverse expression of alpha2A-AR-Cx43 may play key role in inductive and transmission integration underlying mirror image pain conditions.(iii) Expression profiles of spinal alpha2A-AR-Cx43 mediating mirror image painCompared with DRG, spinal alpha2A-AR-Cx43 showed a different pattern. It demonstrated that spinal alpha2A-AR mainly co-localized with astrocyte GFAP and Cx43 in deeper laminar, but few with microglia OX42. The amount of co-localization changed well-fit with time-development of the induction, maintenance and recovery of Bm K I-induced spontaneous pain and mirror mechanical pain hyperalgesia. And, the distribution of spinal alpha2A-AR-Cx43 expression thus showed a migration from medial to lateral. Further qualitative assays revealed that after Bm K I injection, bilateral spinal alpha2A-AR protein and m RNA was decreased, and however Cx43, GFAP and Iba1 was increased. Identically, the reverse effects of pre-treatment with intrathecal clonidine were also clearly found in spinal cord. These results revealed some direct opposite, if any, cellular effects of alpha2A-AR-Cx43 signalings on peripheral detection, transmission and central transfer of pain modulation.(iv) Live cell imaging studies on alpha2A-AR-Cx43 cellular signaling pathwayWith the isolated and acute subcultured spinal astrocytes, the living cell confocal microscopy imaging results verified that neurotransmitter noradrenaline activated and opened gap junctional channels, which allowed fluorescent yellow dye transferring from extracellular bath into the intracellular cytoplasm. Importantly, the noradrenaline-drived dye movement blocked evidently by alpha2A-AR agonist clonidine or gap junction inhibitor CBX, respectively. The results finally provide a strong proof supporting that alpha2A-AR-Cx43 signaling pathways mediated the induction, development, maintenance and recovery of pain.We conclude here that the novel alpha2A-AR-Cx43 signaling pathway is different between peripheral and central induction, transmission, modulation and cellular integration of mirror image pain. The findings would help to elucidate the persistence, chronicalization, diversities, personalization of pain mechanisms, and further provide an more effective anti-pain strategy combing alpha2A-AR and connexins.